A cell is the minimum unit from the viewpoints of functions and structures of organisms. However, an attempt has heretofore been made to investigate, for example, the functions and structures of organisms only for cell populations, but for example, the kinds of substances produced by individual cells have been poorly investigated. The current biological findings are not findings obtained from individual cells but findings obtained from cell populations. Recent investigations have revealed that gene expressions in different cells are diverse even when the cells are obviously similar types (for example, cancer tissues), and it is desired to investigate features, such as gene expression, in each cell.
A human immune system includes T cells, and the T cells include Th2 cells. The Th2 cells are known to produce cytokines, such as IL-4, IL-5, and IL-6. However, previous investigations have not revealed, for example, whether individual cells always produce all of the cytokines at a constant ratio, whether the ratio of the cytokines produced varies according to circumstances, whether cells each producing a part of the cytokines gather to seem to produce all the cytokines when observed as a population, and how the cytokines are produced, and at the same time, what kinds of receptors and transcription factors are produced. Cells are greatly affected by a microenvironment, respond differently by a small number of molecules involved in transcription and translation, and produce probabilistic responses to some extent, and hence expression responses of individual cells are considered to be different from each other.
There are known some methods each involving preparing a double-stranded cDNA using RNA derived from one eukaryotic cell or using a minute amount of RNA that is almost the same as the amount of the RNA derived from one eukaryotic cell, specifically, using about 10 pg of total RNA as a material, and analyzing gene expressions using the cDNA (Non Patent Literature 1 and Non Patent Literature 2). In addition, it is assumed that at a minimum, only one molecule (one copy) of a transcript derived from one cell is present, and hence a device capable of sequencing one nucleic acid molecule has been developed. However, in actuality, the device has the following problems: the device requires use of hundreds of cells, provides low accuracy, and can detect only about 20% of expressed mRNAs.
Accordingly, in order to detect a transcript derived from one cell, it is necessary to increase the amount of the transcript by amplification in advance. As means for amplifying the transcript derived from one cell, there is given means involving solubilizing one cell to separate mRNAs, synthesizing cDNAs using oligo(dT) or random primers, and amplifying the cDNAs by PCR or in vitro transcription (IVT). The use of the PCR has a problem in that not only the amount of the transcript amplified but also the amount of non-specific by-products is large. The cDNAs derived from the mRNAs have various lengths, and it is extremely difficult to amplify fragments having various lengths by PCR without losing a quantification property. In addition, the amount of fragments amplified becomes larger by increasing the number of PCR reaction cycles. However, when a sample includes an extremely high level of cDNAs of a transcript before amplification (for example, tens of thousands of copies are present per cell) and an extremely low level of cDNAs of a transcript (for example, one copy is present per cell), an increase in number of the PCR reaction cycles may lead to a reduction in amplification efficiency of the cDNAs present in a high level. Thus, the presence ratio of the amplified products in all of the resultant amplified products may not reflect the ratio of transcripts in the cell. In addition, IVT has an advantage in that non-specific by-products are produced only in a small amount, but has problems in that cRNA having a length of 1 kb or more is hardly acquired, in that the method requires time and additional work, and in that the amplification amount is not so large. In addition, those methods require treatments for individual cells, and require time and additional work, and hence the number of cells measured is restricted.
As a method of detecting a transcript derived from one cell, there has been proposed a method involving using a bead having bound thereto an oligonucleotide having a barcode sequence (Patent Literature 1). In Patent Literature 1, there is a disclosure of SEQ ID NO: 9 as a specific oligonucleotide bound to a bead, but the oligonucleotide represented by SEQ ID NO: 9 does not have a sequence in which an oligo(dT) is exposed at the 3′ end.